Dynamic Regulation of Class IIa Histone Deacetylases by Phosphorylation

Abstract

Histone deacetylases (HDACs) are critical regulators of host and viral transcription. HDACs carry out their transcriptional repressive activities through substrate deacetylation and participation in numerous multi-protein complexes. Within the HDAC family, class IIa enzymes (HDAC4/5/ 7/9) shuttle between the nucleus and cytoplasm in response to signal-dependent phosphorylations. Within the nucleus, class IIa HDACs interact with transcription factors, co-repressor complexes, and HDAC3 to repress transcription. However, the regulatory roles of numerous additional HDAC phosphorylations remain unexplored, and the functional roles of HDACs outside of the nucleus, during cell cycle progression, and during viral infection remain less poorly understood.

In order to address these remaining questions regarding the function and regulation of class IIa HDACs, this study integrates mass spectrometry-based proteomics, phosphomutant analyses, in vitro enzymatic assays, microscopy, and cell cycle and viral infection studies. We present the first comprehensive protein interactome for HDACs 1-11 in T cells and highlight shared and unique roles for individual HDAC classes. We present a method for assessing protein interaction stabilities using stable isotope labeling in conjunction with a probabilistic scoring method for determination of interaction specificity using class IIa HDACs as a test case.

We further focused our investigation of class IIa HDACs on HDAC5 and identified 14 novel phosphorylations on HDAC5. Generation of serine-to-alanine phosphomutant cell lines for individual sites revealed that phosphorylation of Ser279 within the NLS is important for HDAC5 nuclear localization. We further demonstrate that Ser278 is phosphorylated during mitosis by the kinase Aurora B. Aurora B-mediated phosphorylation of HDAC5 is conserved in HDAC4 and HDAC9, and targets class IIa HDACs to the midzone. During mitosis, HDAC5 exhibits diminished association with co-repressor components and limited in vitro deacetylation activity. Additional analysis of phosphorylations within functional domains of HDAC5 demonstrated that phosphorylation abundances range from ~3-75%, suggesting both dynamic and consitutive modification of individual sites. Together, these results demonstrate that HDAC5 functions are carefully coordinated by post-translational modifications and protein interactions. Finally, we profile the dynamic localization of HDAC5 during HIV-1 infection and accompanying changes in HDAC5 and HDAC3 interactions to gain preliminary insight into class IIa HDAC during viral infection.